**1. Introduction**

Infertility affects up to 15% of couples worldwide, with up to 50% of cases attributable to male factor infertility [1]. In a majority of cases, the precise etiology underlying infertility in the male partner remains unclear. A subset of men with infertility have no sperm in the ejaculate, known as azoospermia, which may further be classified into obstructive (OA) or non-obstructive azoospermia (NOA). The majority of cases of NOA are idiopathic, however some known etiologies include genetic disorders, chemotherapy or radiation, developmental or structural abnormalities, and hormonal imbalances (Table 1). Despite the etiology underlying the spermatogenic dysfunction resulting in NOA, sperm often can be surgically extracted from the testis for use in assisted reproductive technology (ART) with varying success. Intracytoplasmic sperm injection (ICSI) requires only a single spermatozoon for injection into an oocyte, and thus has improved the chances for men with NOA to conceive biological children. In this review, we discuss common etiologies for NOA and the reproductive outcomes for NOA men after surgical sperm retrieval and ICSI.

**Table 1.** Etiologies of non-obstructive azoospermia.


#### **2. Treatment of Non-Obstructive Azoospermia**

Effective managemen<sup>t</sup> of infertility in men with NOA requires testicular sperm retrieval as well as ART in the form of ICSI. Since sperm retrieval involves finding one of

the very limited sites of sperm production within a highly dysfunctional testis of a man with NOA, it is not surprising that the approach used for sperm retrieval can substantially affect the chance of obtaining sperm for fertility.

A wide variety of approaches have been used for attempted sperm retrieval including fine needle aspiration of the testis (testicular sperm aspiration; TESA), random biopsies of testicular tissue to identify foci of sperm production (testicular sperm extraction; "conventional" TESE) as well as directed testicular surgical sperm retrieval using a microsurgical approach (microdissection testicular sperm extraction; microTESE or mTESE).

Each of these methods were compared using a meta-analysis of published literature [2]. Although a recent meta-analysis reported no difference in sperm retrieval rates when comparing conventional TESE to microTESE, it is important to note that this analysis did not require comparative studies so the heterogeneous nature of NOA patients treated at different sites invalidated any meaningful comparison of surgical techniques [3]. The superiority of microTESE is not surprising, as the surgery directs sampling of testicular tissue to the largest seminiferous tubules, which are those most likely to contain sperm [4]. From a laboratory perspective, the microTESE approach is ideal, as it limits the amount of tissue that must be examined by the andrologist to identify sperm to that which is richest in sperm production. Typical search times to find sperm in isolated, dispersed testicular tissue specimens is only 3 to 5 min at experienced centers. Of note, microTESE, although an invasive surgical procedure, has less effect on testicular function than other approaches for sperm retrieval [4].

#### **3. Causes of Non-Obstructive Azoospermia**

NOA occurs secondary to the disruption of spermatogenesis within the testicular parenchyma. This disruption of sperm production is a common phenotype with various underlying etiologies. Although understanding the underlying etiology of azoospermia may help in prognosis and counseling, the precise mechanisms by which spermatogenesis is disrupted in these disorders are not well understood. Men with NOA have varying ranges of spermatogenic failure, and even in 30–60% of those with severely dysfunctional histology (i.e., Sertoli Cell Only (SCO) or maturation arrest) small foci of spermatogenesis can be observed [5]. Furthermore, many other presumed idiopathic cases of NOA are likely to be caused by genetic abnormalities that are ye<sup>t</sup> to be fully delineated.

#### *3.1. Hormonal Imbalances*

Men with hypogonadotropic hypogonadism (HH) suffer from a lack of gonadotropin stimulation, resulting in failure of the testis to produce testosterone or sperm. The defect can be congenital (e.g., Kallmann syndrome, Prader-Willi syndrome) or acquired (e.g., secondary to pituitary tumor or exogenous steroid administration). The resultant phenotype of these men is lack of development of secondary sexual characteristics (with prepubertal phenotype) and infertility. Importantly, because the phenotype is caused by a lack of gonadotropin, treatment of these men with exogenous gonadotropins (e.g., human chorionic gonadotropin (hCG) and recombinant follicle-stimulating hormone (FSH)) can result in the appropriate development of secondary sexual characteristics (i.e., pubic hair development, testis growth, development of muscle mass) and sperm [6]. Men with HH typically do not require ICSI to achieve pregnancy as treatment with exogenous gonadotropins is highly effective in inducing spermatogenesis adequate to allow return of sperm to the ejaculate, which is associated with an increase in endogenous testosterone production [6].

Hyperprolactinemia, or elevated serum prolactin levels, is a rare etiology for azoospermia but clinically relevant. Prolactin is produced by the posterior pituitary and elevated levels can result from a prolactin-secreting adenoma (or prolactinoma) [7]. One study examining prolactin levels in infertile men observed increased prolactin levels in men with asthenozoospermia, oligozoospermia, and azoospermia [8]. Since hyperprolactinemia is typically effectively treated with medical therapy, it is rarely a cause for persistent NOA requiring surgical intervention.

## *3.2. Klinefelter Syndrome*

Klinefelter Syndrome (KS) is the most common sex chromosome aneuploidy in infertile men, with an estimated prevalence of approximately 10% in men with NOA [9]. This syndrome involves the addition of one or more extra X-chromosome(s), resulting most commonly in a 47,XXY karyotype [9–11]. KS is thought to occur secondary to chromosomal nondisjunction during meiosis [12,13]. Physical examination of KS men often reveals characteristic findings of tall stature, reduced testis size, reduced chest and facial hair, gynecomastia, eunuchoid appearance, wide hips, and narrow shoulders [13]. Small testis size is thought to occur due to fibrosis and hyalinization of the seminiferous tubules and is progressive through puberty and adult development [14,15]. Rare, small foci of spermatogenesis in the testes of KS men is hypothesized to be present due to the capability of XXY stem cells to undergo spermatogenesis, or more likely, mitotic errors within the XXY stem cell population resulting in diploid cells capable of completing the remaining spermatogenic process [16].

#### *3.3. Y-Chromosome Microdeletions*

One of the most common identifiable etiologies of NOA are microdeletions of the azoospermia factor (AZF) region of the Y chromosome, and up to 12% of men with NOA harbor AZF microdeletions [17]. There are three loci in the AZF region, which are designated AZFa, AZFb and AZFc, and each locus contains various genes responsible for different aspects of spermatogenesis [18–20]. Microdeletions within AZFc are the most common (up to 80%), whereas AZFa (up to 4%) and AZFb (up to 5%) are less common [21]. Polymerase chain reaction (PCR) is used to detect Y-chromosome microdeletions (YCMDs) as these chromosomal deletions are too small to detect by standard karyotype analysis. Current guidelines recommend testing for YCMD along with karyotype analysis in men with NOA or severe oligozoospermia (<5 million sperm/mL) [22]. Knowing the YCMD status in a man with severe infertility carries important prognostic information as the sperm retrieval rates (SRR) in men with complete AZFa and AZFb deletions is zero, whereas men with AZFc deletions can have SRR of approximately 50–60% [23].

#### *3.4. Malignancy, Chemotherapy, and Radiation*

Malignancy and associated treatments such as chemotherapy and radiation are important causes of azoospermia as approximately 50% of men will be affected by cancer in their lifetime [24]. Chemotherapy targets rapidly dividing cells and testicular germ cells which are mitotically and meiotically active and are highly sensitive to these systemic agents [25]. DNA alkylating agents, as well as platinum-containing chemotherapy agents (such as cisplatin), cross-link DNA and are particularly harmful to spermatogonial stem cells and may result in permanent azoospermia [26,27]. Other chemotherapy agents, such as anthracyclines, antimetabolites, and topoisomerase inhibitors, typically are less gonadotoxic and result only in transient decreases in sperm count because differentiating spermatogonia, and not stem cells, are primarily affected [26]. The CED, or cyclophosphamide-equivalent dose, may be used to determine estimated alkylating agen<sup>t</sup> exposure [28]. One study of adult childhood cancer survivors found that when the CED was less than 4000 mg/m<sup>2</sup> men were normospermic, however there was substantial overlap in the CED values of normozoospermic, oligozoospermic, and azoospermic men [29]. Radiation therapy, on the other hand, may result in irreversible testicular damage secondary to the high radio-sensitivity of testicles [30]. Spermatogonial stem cells are highly sensitive to radiation and are adversely impacted even at low radiation doses (0.1 Gy), with permanent azoospermia typically occurring with doses of 16–20 Gy but reportedly occurring with radiation doses as low as 4 Gy [25,31]. Recovery of spermatogenesis after chemotherapy or radiation therapy depends on the chemotherapy agen<sup>t</sup> used and cumulative chemotherapy or radiation dose [31].

Importantly, as the efficacy of cancer treatments has improved, the number of survivors has increased worldwide [24]. Therefore, strong recommendations from the American Urological Association (AUA), American Society of Clinical Oncology (ASCO), and American Society for Reproductive Medicine (ASRM) have been made to counsel and refer patients for discussion of fertility preservation prior to initiating cancer treatments [32–34]. Additionally, men undergoing chemotherapy or radiation therapy should avoid pregnancy for a minimum of twelve months after completing treatment because of potentially mutagenic effects of the treatments on germ cells [34,35]. Finally, in men with persistent azoospermia after gonadotoxic chemotherapy or radiation treatment, testicular sperm extraction may be performed to harvest sperm for ART [34].
